This proposal focuses on BIN1, the most significant late-onset Alzheimer's disease (LOAD) susceptibility locus identified via genome-wide association studies. AD-associated BIN1 single-nucleotide polymorphisms increase BIN1 expression. Alternative splicing, as well as an increase in BIN1 expression, have also been reported in LOAD. BIN1 is an adaptor protein that regulates membrane dynamics in the context of endocytosis and membrane remodeling. Its function in the brain is not known, and there is much to learn about the relationship between BIN1 and the elevated risk for LOAD. BIN1 can directly bind to Tau, leading to the suggestion that BIN1 might influence tangle pathology in AD. We recently reported predominant BIN1 expression in mature oligodendrocytes in the human and rodent brain. Interestingly, unlike in non-AD brain, neurons in AD brain begin to express BIN1. In preliminary studies, we identified a selective increase in the levels of BIN1 isoform 9 (BIN1iso9) in LOAD. How these observations translate to increased risk for LOAD is not clear. Transgenic mouse models have been invaluable in investigating the disease mechanisms. The overarching goal of this proposal is to test a gain-of-function hypothesis using BIN1 transgenic mice and resolve whether the LOAD- associated changes in BIN1 per se are causal to the disease process or rather they are reactive to the disease. To accomplish this goal, we have generated a transgenic line and used Cre drivers to activate human BIN1iso9 expression in a cell-type specific manner. The proposed investigation will test our central hypothesis that an increase in BIN1iso9 expression will worsen AD-associated pathology and behavior abnormalities. The goal of Aim 1 is to perform targeted proteomics using select reaction monitoring to perform a comprehensive characterization of the BIN1 isoform diversity in LOAD. We will also use immunogold electron microscopy to localize BIN1 with ultrastructural precision in the human brain and AD mouse models. Aim 2 studies will test the hypothesis that transgenic expression of BIN1iso9, which recapitulates BIN1 upregulation in AD, will compromise neuronal endocytosis and synaptic transmission, as well as interfere with myelin protein trafficking in oligodendrocytes. Aim 3 studies will test the hypothesis that BIN1iso9 expression will exacerbate neuropathology and behavioral deficits in mouse models of amyloid and tau pathogenesis. This timely and unique proposal is highly innovative. This investigation using novel BIN1 transgenic mice represents the most direct in vivo approach to rigorously test BIN1's involvement in the biological pathways of AD pathogenesis. In addition, this investigation will use proteomics approaches to clarify altered BIN1 isoform expression in LOAD, precisely define BIN1 localization in the brain, and characterize Bin1 transgenic mice as a resource to the AD field. We believe that successful completion of the proposed investigation will fill significant gaps in our understanding of BIN1 as a risk factor for LOAD, and guide future functional characterization of biological pathways and pathogenic mechanisms regulated by this major LOAD risk gene.